High prevalence of bla CTX‐M‐15 type extended‐spectrum beta‐lactamases in Gambian hooded vultures (Necrosyrtes monachus): A threatened species with substantial human interaction

Abstract One hundred fecal samples from hooded vultures in the Gambia (Banjul area) were investigated for the presence of bacteria with extended‐spectrum cephalosporin‐ (ESBL/AmpC), carbapenemases, and colistin resistance. No Enterobacteriales carrying carbapenemases or resistance against colistin were detected. Fifty‐four ESBL‐producing Escherichia coli and five ESBL‐producing Klebsiella pneumoniae isolates were identified in 52 of the samples, of which 52 E. coli and 4 K. pneumoniae yielded passed sequencing results. Fifty of the E. coli had ESBL phenotype and genotype harboring bla CTX‐M genes, of which 88.5% (n = 46) were the bla CTX‐M‐15 gene, commonly found on the African continent. Furthermore, the genetic context around bla CTX‐M‐15 was similar between isolates, being colocalized with ISKpn19. In contrast, cgMLST analysis of the E. coli harboring ESBL genes revealed a genetic distribution over a large fraction of the currently known existing E. coli populations in the Gambia. Hooded vultures in the Gambia thus have a high ESBL E. coli‐prevalence (>50%) with low diversity regarding key resistance genes. Furthermore, given the urban presence and frequent interactions between hooded vultures and humans, data from this study implies hooded vultures as potential vectors contributing to the further dissemination of antibiotic‐resistance genes.

The hooded vulture (Necrosyrtes monachus) is a small (~70 cm and~2.1 kg) species of vulture, mostly brown, with a wingspan up to 180 cm (BirdLife International, 2022). The species has been listed as critically endangered since 2015 by Birdlife International (BirdLife International, 2021, 2022. Major concerns include intentional and secondary poisoning, hunting, both persecution and unintentional, and loss of habitats (BirdLife International, 2021, 2022Ogada et al., 2012). There have also been reports of vulnerability to avian influenza (BirdLife International, 2021, 2022. Furthermore, vultures are sensitive to diclofenac, and even low levels cause kidney failure and death (Ogada et al., 2012). In the West African region, the decline rate of vultures is high (Henriques et al., 2018), although the coastal zone of Gambia appears to have the largest population estimated at 7000-10,500 in a 600 km 2 sampled area, where it is the most common vulture species in the area (BirdLife International, 2021, 2022Mawdo Jallow et al., 2016). Hooded vultures are at the top of the food web and feed mainly on carrions and insects (BirdLife International, 2021;Henriques et al., 2018). Vultures provide ecosystem services as scavengers contributing to nutrient cycling by consuming organic waste and by competing and thereby controlling other scavengers (Henriques et al., 2018). Thus, vultures may be bioindicators for contaminants, biocides, and other anthropogenic pollution.
Recent reports suggest that over 20% of different vulture species in both Europe (Canary Islands) and Asia carries cefotaximeresistant Escherichia coli isolates (Carvalho et al., 2020)

| Bacterial culturing
At arrival on 22nd Nov 2019, the samples were inoculated in 2 mL brain heart infusion (BHI) broth (Becton Dickinson) with 8 mg/L vancomycin (Sigma-Aldrich), and the broth was incubated for 18-24 h at 36°C in aerobic conditions. For selective screening of cephalosporin-resistant bacteria, 10 µL overnight BHI broth was inoculated on CHROMagar™ C3G R agar (CHROMagar™, France). For selective screening of carbapenem-resistant bacteria, 10 µL overnight BHI broth was inoculated on mSuperCarba™ agar (CHROMagar™, France). To selectively screen putative colistin-resistant bacteria, 10 µL overnight BHI broth was inoculated on Col-APSE agar (CHROMagar™, France). All plates were incubated for 18-24 h at 36°C in aerobic conditions; see Figure 1 for analysis workflow. E. coli CCUG 17620 was used as a negative control for C3G R , mSuperCarba™ and Col-APSE agar plates. K. pneumoniae CCUG 45421 and CCUG 64452 were used as a positive control for C3G R and mSuperCarba™ agar plates, respectively. E. coli CCUG 70662 was used as a positive control for Col-APSE agar plates.

| Isolation and identification
Putative E. coli and K. pneumoniae colonies were isolated from C3G R , mSuperCarba™ and Col-APSE agar. In most cases, one putative colony per plate was selected, although depending on colony morphology, more than one was chosen in some cases.
Identification was performed using matrix-assisted laser

| Genomic analysis
DNA was extracted from all E. coli (n = 54) and K. pneumoniae (n = 5) colonies isolated from C3G agar using MagNA Pure Compact total nucleic acid isolation kit (Roche). Preparation of multiplexed DNA libraries was done using NexteraXT or Nextera DNA Flex library preparation kits (Illumina). The whole genome sequencing was performed using HiSeq. 4000 (Illumina).
Raw reads were trimmed and filtered using CLC genomic workbench version 21.0.4 (Qiagen) with default settings. De novo assembly and QC were performed using CLC genomic workbench version 21.0.4 (Qiagen) with default settings. In silico analysis of multilocus sequence type (MLST) (Larsen et al., 2012) (Chen et al., 2016) and detection of plasmid replicons (Carattoli et al., 2014;Clausen et al., 2018). The prevalence of mobile genetics elements (Johansson et al., 2021) and phylogroup typing (Beghain et al., 2018) was assessed by uploading preassembled fasta files to the Center for Genomic Epidemiology and ClermonTyping (http:// clermontyping.iame-research.center/), respectively. The sequences from the E. coli isolates in this study were compared to all other existing E. coli isolates in Enterobase reported to be collected in Gambia (n = 723) using hierarchical clustering of cgMLST data with the cgMLST V1 + HierCC V1 scheme with NINJA NJ algorithm (Zhou et al., 2018(Zhou et al., , 2020.
No E. coli or K. pneumoniae were isolated from SuperCarba agar.
Seven isolates, four E. coli, and three K. pneumoniae were isolated from Col-APSE agar, but none had phenotypic resistance against colistin when assessed using broth microdilution, and no further analysis was done.
All of the E. coli isolates and the K. pneumoniae isolates from the selective screening for extended-spectrum cephalosporin resistance (n = 52 for E. coli, n = 4 for K. pneumoniae, where three samples contained both E. coli and K. pneumoniae) were defined as multidrugresistant (MDR) having resistance against ≥1 agent in ≥3 different antimicrobial categories (Supporting Information: Table S1 at https://doi.org/10.5281/zenodo.7645125) (Magiorakos et al., 2012 (Carvalho et al., 2020). Blanco et al. also reported lower frequencies (25%-30%) in both griffon and Egyptian vultures breeding in Spain . In comparison, at the time of the study, the prevalence of E. coli resistant to 3rd generation cephalosporins in Spain was 10%-25% ( 3.3 | Plasmid replicons, mobile genetic elements, and genetic context Several different plasmid replicons were identified among the E. coli isolates (Figure 4a). For a few isolates, the genetic context could be identified, but in most cases, not. In isolate 3087, plasmid replicons IncFIB and IncB/O/K/Z were identified in the same genetic context (i.e., same contig) as bla CTX-M-15, qnrS1, tet(A), and ISKpn19 (Figure 4a,b).
For the two E. coli isolates with bla CTX-M-27 , the gene was located on the same contig as IS102 (Figure 4a). A recent systematic review of resistome epidemiology in Africa reports that MGEs in association with ARGs were rarely described in isolates from environmental and animal samples (Sekyere & Reta, 2020). In this study, 84.7% (n = 39/46) of isolates with bla CTX-M-15 , the gene was colocalized with qnrS1 and ISKpn19 (Figure 4a,b). This genetic context has been described by others, including an IncFII bla CTX-M-15harboring plasmid identified from Shigella sonnei in Switzerland (Campos-Madueno et al., 2020). Another study has identified the spread of a novel plasmid containing bla CTX-M-15 together with bla TEM-1 and qnrS1 (Fortini et al., 2015).
The presence of ARB in wild animals is affected by different biological, ecological, and geographic factors, still warranting F I G U R E 3 The minimum spanning tree of hierarchical clustering of cgMLST analysis of 723 Escherichia coli isolates in Enterobase isolated from human (dark blue), primate (light blue), avian (green), and poultry (orange) in Gambia. Red circles indicate ESBL E. coli isolates collected from hooded vultures in this study. further investigations to fully understand dissemination patterns and selection pressure (Arnold et al., 2016;Lee et al., 2020;Mughini-Gras et al., 2019;Wang et al., 2017). Global monitoring through metagenomic analysis of urban sewage revealed a correlation between total AMR abundance and socioeconomic factors, where the highest levels of AMR were found in countries from the African continent (Hendriksen et al., 2019). This has also been shown in a cross-country regression analysis where lower AMR abundance correlated with better infrastructure, access to clean water, and improved sanitation (Laxminarayan et al., 2020).
The data in this study implicate that vultures could act as bioindicators for circulating ARB and ARG in anthropogenicaffected environments. Wild animals living and feeding in human-affected environments are often colonized with ARB of great clinical significance (Dolejska & Literak, 2019;Karesh et al., 2012). Aquatic environments and wastewater treatment plants are often highlighted for the risk of AMR transmission between humans and the environment (Bengtsson-Palme et al., 2019;Calero-Cáceres et al., 2022). This study implies that nonaquatic species such as scavengers and opportunistic feeders in close proximity to anthropogenic environments could pose a risk of AMR transmission between animals, the environment, and humans. In the Gambia, Banjul area, the hooded vultures, apart from feeding from landfills and human waste, are often observed to scavenge close to slaughterhouses and food markets ( Figure 5).
In addition to also having nightly roosting trees in the middle of human settlements, hooded vultures are not only bioindicators for AMR but could also be potential vectors for AMR transmission within different one-health sectors. On the other hand, hooded vultures, as scavengers, remove carcasses that potentially carry pathogenic microorganisms, thereby possibly reducing such a threat to human and animal health. Furthermore, both avian and human pathogenic E. coli strains share a lot of common virulence factors (Kathayat et al., 2021). An interspecies exchange of E. coli strains is, of course, also concerning from a conservation perspective since it might include virulent E. coli strains that could be devastating for a critically endangered species such as the hooded vulture. A recent review investigated the impact of microorganisms on vultures and concluded that their health could be affected by both human pathogenic bacteria and viruses, possibly influencing fitness and mortality (Plaza et al., 2020).
Specifically, hooded vultures infected by avian influenza (H5N1) had neurological symptoms, and white-rumped vultures showed signs of enteritis caused by E. coli (Plaza et al., 2020).

| CONCLUSION
This study has identified a high prevalence of ESBL-producing E. coli among hooded vultures in the Gambia. The isolates identified were distributed over a large fraction of the currently known genetic diversity of the existing E. coli population in the Gambia collected from human, primate, and avian sources. However, there was a low diversity regarding ESBL genes with bla CTX-M-15 colocalized with ISKpn19 dominating across different STs, and no carbapenemases were found. Based on data from this report, hooded vultures, as scavengers and top predators, may function as bioindicators for the presence of ARB in relation to anthropogenic and modified environments. Furthermore, this study highlights how exposed vultures are to anthropogenic impact, not only posing a threat to the birds but also as they may serve as vectors for further dissemination of ARB in the environment and (re-)transmission of ARB to humans. Future research should focus on possible mitigation strategies to reduce the risk of AMR dissemination by a hooded vulture, with special attention on how to minimize bird interaction with potential point sources of AMR and areas where the risk of AMR transmission between birds and humans is high. The conservation aspect of hooded vultures must be given particular attention when forming mitigation strategies for AMR transmission between different One Health sectors since the vultures are not only at risk of being vectors for AMR transmission but also pose a risk of being exposed to harmful zoonotic pathogens.
F I G U R E 5 Hooded vultures scavenging nearby and in slaughterhouses show anthropogenic interaction posing a risk for further dissemination in the environment and further transmission to humans and animals (photograph taken by F. Camara).